Genetic identity and differential expression of p38.5 (Haymaker) in human malignant and nonmalignant cells

Previous studies from our laboratory revealed a novel protein of 38.5 kD on the surface of malignant cell lines of hematopoetic origin that exhibit su sceptibility to naive natural killer (NK) cell-mediated lysis. In contrast, p38.5 was not detected on the surface of NK cell-resistant carcinoma cell lines or normal cells. We now report that this protein is differentially ex pressed, intracellularly, in malignant cell lines of both hematopoetic and epithelial origin compared with nonmalignant cells. To characterize p38.5 f urther, we used a previously developed antipeptide antibody (anti-II-mer) t o probe cDNA expression libraries and subsequently performed 5' extension b y rapid amplification of cDNA ends (RACE). Sequence analyses of these cDNA clones reveal open reading frames (ORFs) that include the previously identi fied I I-mer peptide from purified, native p38.5 and that have identical se quences to a gene of unknown function on chromosome 19. Nucleotide sequence data obtained from these cDNA clones, as well as analysis of the genomic s equence, permitted design of primers for reverse transcriptase-polymerase c hain reaction (RT-PCR) that resulted in a cDNA clone encoding an ORF of 361 amino acids; the clone was identical to a sequence encoded by an unpublish ed mRNA in GenBank. Anti-p38.5 antibody against the II-mer peptide encoded in exon 5 and against a 25-mer peptide encoded in exon I both reacted with the same protein in immunoprecipitation studies, providing further evidence of identity. RT-PCR and Northern blot analyses both demonstrated p38.5 gen e transcripts in normal cells, nonmalignant cell lines and malignant cell l ines of epithelial as well as hematopoietic origin. Semiquantitative studie s revealed a greater level of p38.5 gene transcription in malignant cell li nes compared with nonmalignant cells. Immunoblot analyses of protein expres sion confirmed and extended the latter studies by revealing substantially g reater levels of the 38.5 kD protein in whole cell extracts of malignant ce ll lines compared with nonmalignant cells. Quantitative differences in dete ction of the 38.5 kD protein and mRNA in NK susceptible- hematopoietic mali gnancies compared with NK resistant-carcinomas were not observed. These exp eriments suggest that the p38.5 gene (Haymaker) is widely expressed in huma n cells of different tissue origins but that elevated expression is associa ted with the malignant phenotype. (C) 2001 Wiley-Liss, Inc.